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Fig. 3. Development of mouse metanephroi in whole-organ cultures and its
modulation by Sema4d. (A-C) Phase-contrast images of a kidney
rudiment isolated at E12 (A) and of the same kidney after 24 hours (B) and 48
hours (C) in culture. (D-F) Maximal projections of confocal images of
whole cultured metanephroi showing the ureteric tree stained with an
anti-calbindin antibody (green, D) and the condensing mesenchyme stained with
an anti-WT1 antibody (red, E,F) at E12 (D) and after 24 hours (E) and 48 hours
(F) in culture. UT, ureteric tip; CD, collecting duct; MM, metanephrogenic
mesenchyme; CB, comma-shape bodies, two examples are indicated by the double
arrow. (G) Illustration of the method used for quantifying ureteric
branch points (bp) and ureteric tips (arrows, UT) in maximal confocal
projections. (H,I) Typical examples of E12 metanephroi cultured
for 24 hours in medium supplemented with concentrated medium of
mock-transfected HEK293T cells (H) or Sema4d-AP-transfected HEK293T cells (I).
The ureteric tree is labelled with an anti-calbindin antibody (green).
(J) Western blot analysis demonstrating the efficacy of Sema4d in
inducing RhoA activation in a GST-RBD-based assay in HEK293T cells transfected
with plexin B1 (PlxnB1) and PDZRhoGEF (PRG). Anti-Myc-positive bands in
western blotting serve as loading controls. (K) Sema4d treatment
reduces the number of ureteric tips and branch points over mock treatment in
sister cultures of metanephroi. (L) Sema4d treatment decreases the size
of the developing kidney over mock treatment. Only sister kidneys cultured for
48 hours were taken for analysis. *P<0.001 in
comparison with mock, Student's paired t-test. Scale bars: 200 µm
in A-I.
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